Hdl-associated protein extraction and detection

ABSTRACT

Provided herein are compositions, systems, and methods for extracting and detecting at least one HDL-associated protein (e.g., ApoA1) from a sample (e.g., plasma or serum sample). In certain embodiments, a strong organic acid and hydrophilic organic solvent are mixed with the sample; after centrifugation, the supernatant is transferred to a second container where it is mixed with a non-polar organic solvent; after centrifugation, the lower aqueous layer is transferred to a third container; and then at least a portion of the transferred aqueous layer is subjected to a detection assay such that at least one HDL-associated protein is detected.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority benefit from U.S. ProvisionalPatent Application 62/052,854, filed Sep. 19, 2014, which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are compositions, systems, and methods for extractingand detecting at least one HDL-associated protein (e.g., ApoA1) from asample (e.g., plasma or serum sample). In certain embodiments, a strongorganic acid and hydrophilic organic solvent are mixed with the sample;after centrifugation, the supernatant is transferred to a secondcontainer where it is mixed with a non-polar organic solvent; aftercentrifugation, the lower aqueous layer is transferred to a thirdcontainer; and then at least a portion of the transferred aqueous layeris subjected to a detection assay such that at least one HDL-associatedprotein is detected.

BACKGROUND

Serum lipoproteins comprise a heterogeneous population of lipid-proteincomplexes that can be grouped into broad classes, very low (VLDL), low(LDL) and high (HDL) density, based on differences in particle densityrelated to lipid and protein content. VLDL and LDL are composed ofpredominately lipid, while high density lipoproteins have a highercontent of protein (about 50%). The density of LDL is between1.006-1.063 g/ml while that of HDL and HDL-like particles is 1.063-1.21g/ml. Classical methods to separate HDL from VLDL and LDL employsequential density ultracentrifugation using potassium bromide saltsolutions prepared with densities in the range of each lipoproteinclass. One drawback of these methods for the preparation of purified HDLis that they require a minimum of two prolonged ultracentrifugationsteps. The first step, which isolates VLDL and LDL from HDL, requires an18 hour ultracentrifugation spin in d=1.063 g/ml KBr salt solution. Thebuoyant VLDL and LDL are concentrated in the upper layers of the saltgradient and can be easily removed leaving the less buoyant HDL alongwith other heavier proteins concentrated in the bottom layers. The HDLis then separated from other lipid-free serum proteins by performing asecond ultracentrifugation step for 21 hours in d=1.21 g/ml KBr saltsolution. The HDL is buoyant in this density salt solution thus at theend of the centrifugation, the upper layers of the gradient containsprimarily HDL leaving other plasma proteins in the bottom fraction. Thissequential density gradient ultracentrifugation procedure is the “goldstandard” for isolation of HDL. However the prolonged time required forboth ultracentrifugation steps and the need for multiple densityadjustments clearly limits the throughput of the procedure.

SUMMARY OF THE INVENTION

Provided herein are compositions, systems, and methods for extractingand detecting at least one HDL-associated protein (e.g., ApoA1) from asample (e.g., plasma or serum sample). In certain embodiments, a strongorganic acid and hydrophilic organic solvent are mixed with the sample;after centrifugation, the supernatant is transferred to a secondcontainer where it is mixed with a non-polar organic solvent; aftercentrifugation, the lower aqueous layer is transferred to a thirdcontainer; and then at least a portion of the transferred aqueous layeris subjected to a detection assay such that at least one HDL-associatedprotein is detected.

In some embodiments, provided herein are methods of extracting anddetecting at least one HDL-associated protein comprising: a) mixing, ina first container, a separating solution and a serum or plasma sample togenerate a first mixed sample, wherein the separating solution comprisesa strong organic acid and a hydrophilic organic solvent, and wherein theseparating solution makes up greater than 50% of the first mixed sample;b) exposing the first mixed sample to centrifugal force such that thefirst mixed sample separates into a pellet and supernatant; c)transferring at least a portion of the supernatant to a second containersuch that it is separated from the pellet; d) adding a non-polar organicsolvent to the second container at a ratio of greater than 1:1 togenerate a second mixed sample; e) exposing the second mixed sample tocentrifugal force such that the second mixed sample separates into anupper organic solvent layer and a bottom aqueous layer; f) transferringat least a portion of the bottom aqueous layer to a third container suchthat it is separated from the upper organic solvent layer; and g)subjecting at least a portion of the bottom aqueous layer to a detectionassay such that at least one HDL-associated protein is detected.

In additional embodiments, the at least one HDL-associated protein islisted in Table 1. In further embodiments, the at least oneHDL-associated protein is human ApoA1. In other embodiments, the mixingin step a) is with a serum sample. In other embodiments, the mixing instep a) is with a plasma sample. In further embodiments, the strong acidis selected from the group consisting of: trifluoroacetic acid, formicacid, acetic acid, pentafluoroproprionic acid, and heptafluorobutryicacid. In other embodiments, the hydrophilic organic solvent is selectedfrom the group consisting of: acetonitrile, methanol, ethanol, propanol,isopropanol, butanol, and tetrahydrofuran. In additional embodiments,the non-polar organic solvent is selected from the group consisting of:hexane, heptane, octane, cyclohexane, methylcyclohexane, and mixturesthereof. In certain embodiments, the separating solution makes upgreater than 55% of the first mixed sample (e.g., greater than 60% . . .75% . . . 87% . . . 93% or more). In other embodiments, the non-polarorganic solvent is added to the second container at a ratio of at least2:1. In other embodiments, the methods further comprise adding aprotease and internal standard to the bottom aqueous layer prior to thesubjecting to the detection assay.

In some embodiments, the detection assay comprises mass spectrometry. Inparticular embodiments, the detection assay comprises liquidchromatography. In other embodiments, the detection assay comprisesLC-MRM-MS. The present invention is not limited by the methods used todetect HDL-associated proteins. In certain embodiments, the detectionmethod is selected from the following: mass spectrometry, surfaceplasmon resonance, an in vitro assay, an activity assay,co-immunoprecipitation assay, mass spectrometry, Fluorescence EnergyTransfer (FRET), bioluminescence energy transfer (BRET), an immunoassay(e.g., ELISA), interferometry, Biolayer Interferometry (BLI), DualPolarization Interferometry (“DPI”), Ellipsometry, and Quartz CrystalMicrobalance (see, e.g., U.S. Pat. Pub. 20130017556, herein incorporatedby reference in its entirety).

In particular embodiments, provided herein are methods comprising: a)mixing a sample with a buffer solution and a pH sensitive protease togenerate a mixture, wherein the sample comprises a purified protein, andwherein the pH of the buffer solution changes based on temperature; b)incubating the mixture at a first temperature that causes the buffer tohave a first pH, wherein the first pH is in the optimum activity rangeof the pH sensitive protease such that the pH sensitive protease digeststhe purified protein generating protein fragments; c) incubating themixture at a second temperature that causes the buffer to have a secondpH, wherein the second pH is outside the optimum activity range of thepH sensitive protease thereby reducing the activity of the pH sensitiveprotease; and d) subjecting the mixture to a detection method comprisingmass spectrometry such that the peptide fragments are detected.

In particular embodiments, the pH sensitive protease is Trypsin, LysC,GluC, ArgC, AspN, Chymotrypsin, Pepsin. In additional embodiments, thepurified protein comprises human ApoA1 or other HDL-associated protein.In certain embodiments, the first temperature is about 37 degreesCelsius (e.g., 31-45 degrees Celsius). In other embodiments, the secondtemperature is about 4 degrees Celsius (e.g., 0-8 degrees Celsius). Insome embodiments, the detection method comprises LC-MRM-MS.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary embodiment of an HDL-associated proteinextraction and detection protocol.

FIG. 2 shows an additional exemplary embodiment of an HDL-associatedprotein extraction and detection protocol that employs LysC digestion ofApoA1, addition of internal standards, and LC-MRM-MS detection.

FIGS. 3A-D show the improvement of ApoA-I digestion using thepH-sensitive buffer system described herein.

DEFINITIONS

As used herein, “high density lipoprotein” or “HDL” is a circulating,non-covalent assembly of amphipathic proteins that enable lipids likecholesterol and triglycerides to be transported within the water-basedbloodstream. HDL is composed of about 50% by mass amphipathic proteinsthat stabilize lipid emulsions composed of a phospholipid monolayer(about 25%) embedded with free cholesterol (about 4%) and a core oftriglycerides (about 3%) and cholesterol esters (about 12%). Subclassesof HDL include HDL2 and HDL3. HDL2 particles are larger and contain ahigher content of lipid whereas HDL3 particles are smaller and containless lipid. Further subclasses include from largest particle to smallestparticle, HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c.

As used herein, a “lipoprotein” refers to a type of protein to which oneor more lipid molecules is attached or is capable of being attached. Insome cases, a lipoprotein may be a “lipid-poor lipoprotein” in whichfour or fewer molecules of phospholipid are bound. As used herein, alipoprotein includes a protein to which no lipid is attached but whichcan be exchanged in an HDL particle (e.g. an apolipoprotein).

As used herein, “sample” refers to a portion of a larger whole to betested. A sample includes but is not limited to a body fluid such asblood, cerebral spinal fluid, urine, saliva, plasma, serum, and thelike.

As used herein, “blood sample” refers to refers to a whole blood sampleor a plasma or serum fraction derived therefrom. In certain embodiment,a blood sample refers to a human blood sample such as whole blood or aplasma or serum fraction derived therefrom. In some embodiments, a bloodsample refers to a non-human mammalian (“animal”) blood sample such aswhole blood or a plasma or serum fraction derived therefrom.

As used herein, the term “whole blood” refers to a blood sample that hasnot been fractionated and contains both cellular and fluid components.

As used herein, “plasma” refers to the fluid, non-cellular component ofthe whole blood. Depending on the separation method used, plasma may becompletely free of cellular components, or may contain various amountsof platelets and/or a small amount of other cellular components. Becauseplasma includes various clotting factors such as fibrinogen, the term“plasma” is distinguished from “serum” as set forth below.

As used herein, the term “serum” refers to whole mammalian serum, suchas, for example, whole human serum, whole serum derived from a testanimal, whole serum derived from a pet, whole serum derived fromlivestock, etc. Further, as used herein, “serum” refers to blood plasmafrom which clotting factors (e.g., fibrinogen) have been removed.

As used herein, the term “strong organic acid” refers to an acid whichcompletely or substantially dissociates in aqueous solution. Stateddifferently, a strong organic acid is one which has an acidity constant,Ka, of more than about 1×10⁻². The strength of an acid HA in a solvent Sis usually defined as being proportional to its acidity constant, i.e.,the equilibrium constant Ka for the equilibrium:

$\begin{matrix}\left. {{HA} + S}\rightleftarrows{A^{-} + {SH}^{\; +}} \right. & (9) \\{K_{a} = \frac{\left\lbrack A^{-} \right\rbrack \left\lbrack {SH}^{\; +} \right\rbrack}{\lbrack{HA}\rbrack}} & (10)\end{matrix}$

In equation (10), the constant concentration of the solvent is includedin the value for Ka. Since the acidity constant is the ratio of ionizedto unionized species, the higher the Ka for a particular organic acid,the greater the extent of the ionization (in a particular solventsystem) and the stronger the acid. Examples of such acids include, butare not limited to: trifluoroacetic acid, formic acid, acetic acid,pentafluoroproprionic acid, and heptafluorobutryic acid.

DETAILED DESCRIPTION

Provided herein are compositions, systems, and methods for extractingand detecting at least one HDL-associated protein from a sample (e.g.,plasma or serum sample). In certain embodiments, a strong organic acidand hydrophilic organic solvent are mixed with the sample; aftercentrifugation, the supernatant is transferred to a second containerwhere it is mixed with a non-polar organic solvent; aftercentrifugation, the lower aqueous layer is transferred to a thirdcontainer; and then at least a portion of the transferred aqueous layeris subjected to a detection assay such that at least one HDL-associatedprotein is detected.

In certain embodiments, the purification methods described herein areemployed to purify human ApoA1, and particular modified amino acids inApoA1 (Tyr192 or Trp72) are detected. The myeloperoxidase (MPO)-drivenchlorination of apolipoprotein A-I tyrosine 192 (Tyr192) has been shownto be elevated in the presence of vessel wall inflammation, and mayserve as a specific biomarker for inflammation associated withcardiometabolic disease (see, Bergt et al. (2004) Proc. Nat. Acad. Sci.U.S.A., 101, 13032-13037; and Shao et al. (2011) J. Biol. Chem., 287,6375-63861; both of which are herein incorporated by reference).Inflammation mediated oxidation of apolipoprotein A-I has also beenshown in the modification of tryptophan 72 (Trp72) to2-hydroxytryptophan, a.k.a. oxindolylalanine (Oia72) (Huang et al.(2014) Nature Med., 20, 193-203, herein incorporated by reference).Prior work in the field indicates that the modified amino acid residuesare present in circulating plasma amount at four to five orders ofmagnitude less than their unmodified counterparts, presenting achallenge in both detection and quantification. The methods andcompositions described herein may be used to quantify low levels ofchlorinated Tyr192 (Cl-Tyr192) and Oia72 in apolipoprotein A I relativeto amounts of unmodified Tyr192 and Trp72.

Previously, total levels of Cl-Tyr were determined by isolating HDL bysequential ultracentrifugation, delipidating the HDL particles, reducingall of the protein to its amino acid constituents using acid hydrolysis,and quantifying total Cl-Tyr using isotope dilution GC-MS (see, Bergt etal.). Subsequent methodology specifically quantified individual tyrosineresidues from ApoA-I by digesting HDL protein with trypsin andquantifying relative amounts of unmodified and modified tyrosineresidues from ApoA-I by selected reaction monitoring (SRM) of theirtryptic peptides using isotope dilution LC-MS (see, Shao et al.). Thedetection of ApoA-I Oia72 has previously been accomplished by antibodyrecognition of HDL isolated from atherosclerotic lesions (see, Huang etal.). Such prior work relies on ultracentrifugation for the isolation ofHDL (of which ApoA-I is a major component). Such a method is limited inits capacity to prepare multiple samples in parallel and is timeconsuming, taking several hours to complete. The use of high amounts ofsalts and sucrose require that the isolated HDL be further processedbefore it is in a solution suitable for enzymatic digestion (i.e.trypsin). The use of trypsin as the digesting enzyme on ApoA-I yieldsabundant missed cleavage fragments that may all contain the targetedresidue (i.e, Y192), splitting the signal for a given chemical targetamongst several peptide products and having a detrimental effect onassay sensitivity.

FIGS. 1 and 2 show exemplary embodiments of the protein extraction anddetection protocol of the present invention. A patient serum or plasmasample is precipitated with the addition of a strong organic acid (e.g.,0.1% trifluoroacetic acid (TFA)) in a hydrophilic organic solvent (e.g.,1-propanol) at a ratio of, for example, 2 to 1. This step depletes theserum of large, abundant proteins (i.e. serum albumin, transferrin,etc.) that could interfere with detection of target peptides downstream.The precipitation is facilitated by brief mixing, after which theprecipitate mixture is centrifuged and the supernatant transferred to anew tube. A non-polar organic solvent (e.g., cyclohexane) is added tothe hydrophilic organic solvent extract at, for example, a 2:1 ratio,and briefly mixed. The hydrophilic organic solvent and strong organicacid (e.g., 1-propanol and TFA) from the prior extraction are misciblein the non-polar organic solvent (e.g., cyclohexane), while water isimmiscible, forming an interface when the mixture is allowed to settle(accelerated by centrifugation). The bottom aqueous layer containingApoA-I and other HDL associated proteins is then transferred to a newtube. The HDL-associated proteins can then be detected by anyappropriate method.

The extraction method described herein can be performed in minutes toprepare a patient sample for enzymatic digest in which ApoA-I is themost abundant protein present and large abundant serum proteins such asserum albumin are not present. The extraction method also can be readilyperformed in a parallel fashion (i.e. 96-well plates).

The following additional steps, found in FIG. 2, may also be used.Combine the sample 1:1 with a 2× concentrated digestion buffer (e.g.,100 mM Tris-HCl, pH 9.0 (25° C.); 50% methanol) yields the final samplein 1× digest buffer (50 mM Tris-HCl, pH 9.0 (25° C.); 25% methanol).LysC is added to the sample at an approximately 1:20 wt/wt enzyme tosubstrate ratio (where substrate is protein in the sample) in additionto stable isotope labelled internal standard peptides correlating to theendogenous target peptides for modified and unmodified Tyr192 and Trp72.The sample is then digested at 37° C. and halted by cooling to 4° C., atwhich point the digested samples are submitted for LC-MRM-MS allowingfor the specific detection and quantification of the target peptides.This solution targets two products of inflammation mediated oxidation:chlorinated tyrosine 192 and 2-hydroxy tryptophan 72 in ApolipoproteinA-I. The modified peptides are quantified relative to their correlatedunmodified peptides. LysC is used as the enzyme to produce peptideproducts with no significant missed cleavage products associated withthe target amino acid residues. The sequence of the tyrosine 192containing peptide is:

Glu-Asn-Gly-Gly-Ala-Arg-Leu-Ala-Glu-Tyr/Cl-Tyr-His-Ala-Lys (SEQ ID NO:1)

The sequence of the tryptophan containing peptide is:

Leu-Arg-Glu-Gln-Leu-Gly-Pro-Val-Thr-Gln-Glu-Phe-Trp/Oia-Asp-Asn-Leu-Glu-Lys(SEQ ID NO:2).

This protocol uses a specific digestion buffer system to optimize digestpH and peptide/internal standard solubility. In a Tris buffer system,the pH changes with temperature. This solution utilizes a Tris bufferwith a pH of 9.00 at 25° C. At 37° C., the pH is 8.70, which is in theoptimum range for LysC (pH 8.5-8.8). At 4° C., the pH of the bufferincreases to 9.56, which in addition to the decreased temperature is nonideal for the enzyme. The high pH in addition to the 25% methanol in thebuffer is optimal to maintain peptide solubility and stability for theTrp72/Oia72 peptides, which are optimum under basic conditions with atleast 20% organic content.

The above protocol may employ synthetic peptide internal standards, ofwhich those internal standards correlating to the modified peptidetargets (Cl-Tyr192 and Oia72) fully incorporate the desired modificationwhen synthesized, as opposed to performing chemical modifications tounmodified peptide or protein which can result in variation desiredproduct formation.

Using the method in FIG. 2, ApoA-I is rapidly prepared from patientserum/plasma samples in minutes for digestion. The resulting extract ismostly ApoA-I and other HDL-associated proteins, and large, abundantproteins that could interfere with downstream analysis are not present.Digestion with LysC is rapid. With the use of the digestion buffercomposition, target peptides are produced at approximately the same rateand reach maximum abundance after about one hour of digestion.Endogenous and stable isotope labelled internal standard peptidescontaining the target modified and unmodified amino acid residues arereadily detected and quantified over 5 orders of magnitude of dynamicrange.

The precipitation and extraction procedure outlined in FIG. 2 yields asample composed of mostly ApoA-I and other HDL-associated proteins.Large, abundant proteins that may suppress ApoA-I signal (i.e. serumalbumin) are in low abundance or not detected. (See Table 1)

TABLE 1 Identified Proteins from TFA/Propanol Extracted Serum SequenceMol. weight Posterior Error HDL Protein names Gene names Peptidescoverage (%) [kDa] Probability Intensity Associated? Apolipoprotein A-IAPOA1 15 64 30.777  6.75E−242 1.77E+10 ✓ 1 Apolipoproteln A-II APOA2 764 11.175 1.11E−68 5.50E+09 ✓ 1 Alpha-1-antitrypsin SERPINA1 20 54.146.736 0.00E+00 1.86E+09 ✓ 1 Apolipoprotein C-III APOC3 3 53.5 10.8523.14E−28 1.62E+09 ✓ 1 Apolipoprotein C-I APOC1 4 46.8 8.647 3.11E−231.43E+09 ✓ 1 Apolipoprotein A-IV APOA4 15 55.8 45.398  5.51E−2181.28E+09 ✓ 1 Apolipoprotein C-II APOC2 5 56.4 11.284 1.02E−45 9.44E+08 ✓1 Angiotensinogen AGT 6 25.2 53.154 3.89E−62 2.74E+08 ✓ 1 Complement C3C3 9 9.4 187.15  2.85E−118 1.40E+08 ✓ 1 Complement C4-B C48 9 6.9 187.67 2.27E−112 8.90E+07 ✓ 1 Serum amyloid A-4 protein SAA4 2 16.2 14.7462.28E−05 4.86E+07 ✓ 1 Transthyretin TTR 3 37.4 15.887 1.44E−50 4.22E+07✓ 1 Pigment epithelium-derived factor SERPINF1 10 35.4 46.312 2.51E−593.73E+07 ✓ 1 Apolipoprotein L1 APOL1 9 31.3 42.158 7.86E−43 3.68E+07 ✓ 1Apolipoprotein F APOF 2 10.1 33.463 4.13E−15 3.21E+07 ✓ 1 Hemoglobinsubunit beta HBB 4 30.6 15.998 3.17E−54 2.97E+07 X 0 Apolipoprotein MAPOM 2 21.8 21.253 1.32E−14 2.71E+07 ✓ 1 Hemoglobin subunit alpha HBA1 345.8 15.257 2.07E−16 2.62E+07 X 0 Apolipoprotein C1 APOC1 2 29.6 5.83481.67E−07 2.48E+07 ✓ 1 Fibrinogen alpha chain FGA 2 5.6 69.756 9.67E−602.08E+07 ✓ 1 Apolipoprotein E APOE 5 27.1 36.154 9.83E−55 1.52E+07 ✓ 1Inter-alpha-trypsin Inhibitor heavy chain H4 ITIH4 3 4.6 79.952 2.49E−171.10E+07 ✓ 1 Serum amyloid A-1 protein SAA1 1 10.7 13.532 5.96E−041.06E+07 ✓ 1 Alpha-2-antiplasmin SERPINF2 7 18.7 54.565 4.09E−528.41E+06 X 0 Platelet basic proteinactivating peptide 2 PPBP 3 21.113.894 2.46E−10 7.88E+06 X 0 Thyroxine-binding globulin SERPINA7 5 16.146.324 4.10E−42 6.95E+06 X 0 Alpha-2-HS-glycoprotein AHSG 1 2.7 39.3249.55E−06 3.75E+06 ✓ 1 Complement C4-A C4A 9 6.9 187.7  1.16E−1063.71E+06 ✓ 1 Beta-2-microglobulin B2M 3 25.2 13.714 2.18E−13 2.77E+06 X0 Complement factor D CFD 1 6.7 27.033 1.33E−14 2.61E+06 X 0 SerglycinSRGN 1 8.2 17.652 5.79E−09 2.37E+06 X 0 Retinol-binding protein 4 RBP4 211.1 22.944 1.50E−06 1.87E+06 ✓ 1 Alpha-2-macroglobulin A2M 1 20 9.75231.26E−04 1.36E+06 X 0 Apolipoprotein D APOD 1 5.8 21.275 1.21E−041.36E+06 ✓ 1Sample preparation is more easily accomplished in parallel using theextraction method. It can be performed in multi-well plates andautomated by liquid handling robot. Ultracentrifugation severely limitsthe number of samples that can be simultaneously prepared.

As described above, the extraction protocol may employ LysylEndopeptidase (LysC) as the enzyme for digestion of protein intopeptides. Digestion with LysC produces single peptides containing targetamino acids versus trypsin which produces multiple abundant missedcleavage products that impact sensitivity of measurement. Enzymaticdigestion using trypsin is capable of producing multiple missed cleavageproducts—peptides that contain the target amino acid but with varyingtermini based on enzyme inefficiencies and limitations. For instance,trypsin digestion of ApoA-I could readily yield 6 peptides that includeTyr192, meaning that one would have to quantify 24 peptide targets toaccount for Cl-Tyr192, Tyr192, and their corresponding internalstandards. Using LysC to digest ApoA-I yields 1 peptide product with nodetectable missed cleavage product. The use of LysC also significantlyreduces the amount of time to generate a maximum peptide yield(approximately 1 hour) when compared to trypsin (greater than 18 hours).

Use of temperature sensitive buffer with some organic solvent content(50 mM Tris-HCl, pH 9.0 (25° C.), 25% Methanol) optimizes pH for digestat 37° in addition to helping hinder enzyme activity at 4° andstabilizing target peptide solubility. Compared to a normal digestionbuffer (50 mM Tris, pH 7.8 in Water) (FIGS. 3A and 3B), the targetpeptides are generated at correlated rates (FIG. 3D) and reach a maximumpeptide yield after approximately 1 hour of digestion (FIG. 3C).

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A method of extracting and detecting at least oneHDL-associated protein comprising: a) mixing, in a first container, aseparating solution and a serum or plasma sample to generate a firstmixed sample, wherein said separating solution comprises a strongorganic acid and a hydrophilic organic solvent, and wherein saidseparating solution makes up greater than 50% of said first mixedsample; b) exposing said first mixed sample to centrifugal force suchthat said first mixed sample separates into a pellet and supernatant; c)transferring at least a portion of said supernatant to a secondcontainer such that it is separated from said pellet; d) adding anon-polar organic solvent to said second container at a ratio of greaterthan 1:1 to generate a second mixed sample; e) exposing said secondmixed sample to centrifugal force such that said second mixed sampleseparates into an upper organic solvent layer and a bottom aqueouslayer; f) transferring at least a portion of said bottom aqueous layerto a third container such that it is separated from said upper organicsolvent layer; and g) subjecting at least a portion of said bottomaqueous layer to a detection assay such that at least one HDL-associatedprotein is detected.
 2. The method of claim 1, wherein said at least oneHDL-associated protein is listed in Table
 1. 3. The method of claim 1,wherein said at least one HDL-associated protein is human ApoA1.
 4. Themethod of claim 1, wherein said mixing in step a) is with a serumsample.
 5. The method of claim 1, wherein said mixing in step a) is witha plasma sample.
 6. The method of claim 1, wherein said strong acid isselected from the group consisting of: trifluoroacetic acid, formicacid, acetic acid, pentafluoroproprionic acid, and heptafluorobutryicacid.
 7. The method of claim 1, wherein said hydrophilic organic solventis selected from the group consisting of: acetonitrile, methanol,ethanol, propanol, isopropanol, butanol, acetone, and 1,4 dixoane, andtetrahydrofuran.
 8. The method of claim 1, wherein said non-polarorganic solvent is selected from the group consisting of: hexane,heptane, octane, cyclohexane, methylcyclohexane, and mixtures thereof.9. The method of claim 1, wherein said separating solution makes upgreater than 75% of said first mixed sample.
 10. The method of claim 1,wherein said non-polar organic solvent is added to said second containerat a ratio of at least 2:1.
 11. The method of claim 1, wherein saiddetection assay comprises mass spectrometry.
 12. The method of claim 1,wherein said detection assay comprises liquid chromatography.
 13. Themethod of claim 1, wherein said detection assay comprises LC-MRM-MS. 14.The method of claim 1, further comprising adding a protease and internalstandard to said bottom aqueous layer prior to said subjecting to saiddetection assay.
 15. A method comprising: a) mixing a sample with abuffer solution and a pH sensitive protease to generate a mixture,wherein said sample comprises a purified protein, and wherein the pH ofsaid buffer solution changes based on temperature; b) incubating saidmixture at a first temperature that causes said buffer to have a firstpH, wherein said first pH is in the optimum activity range of said pHsensitive protease such that said pH sensitive protease digests saidpurified protein generating protein fragments; c) incubating saidmixture at a second temperature that causes said buffer to have a secondpH, wherein said second pH is outside said optimum activity range ofsaid pH sensitive protease thereby reducing the activity of said pHsensitive protease; and d) subjecting said mixture to a detection methodcomprising mass spectrometry such that said peptide fragments aredetected.
 16. The method of claim 15, wherein said pH sensitive proteaseis LysC.
 17. The method of claim 15, wherein said purified proteincomprises human ApoA1.
 18. The method of claim 15, wherein said firsttemperature is about 30-45 degrees Celsius.
 19. The method of claim 15,wherein said second temperature is about 0-8 degrees Celsius.
 20. Themethod of claim 15, wherein said detection method comprises LC-MRM-MS.